Cervical cancer incidence in the US has been dramatically reduced as a result of secondary prevention efforts in the form of successful screening programs. Prophylactic HPV vaccines now offer a method for primary prevention, but since they target only a limited number of HPV types that cause cervical cancer, screening will still be required even in vaccinated cohorts. However, current screening algorithms are expected to be increasingly inefficient as the disease prevalence declines as a result of effective vaccination in large populations. Currently, management of screen-positive women is dictated by lesion grade determined by histomorphologic interpretation of a diagnostic biopsy. While low-grade SIL (LSIL [CIN 1]) is managed conservatively with repeat screening, high-grade SIL 3 (HSIL [CIN 3]) requires surgical excision. Grade 2 SIL (HSIL 2 [CIN 2]), that shares features of both LSIL and HSIL 3, demonstrates poor interobserver reproducibility. This diagnosis commonly leads to an excision (although the current management guidelines provide conservative management with follow-up as an option as well). The limitations in the accuracy of histologic grading of HSIL 2 (CIN 2) can result in overtreatment of lesions inappropriately labeled high grade, potentially causing unnecessary morbidity, or less commonly, a failure to treat lesions that may progress. Diagnostic markers to add to the morphologic assessment are urgently needed to improve the specificity and positive predictive value of the cervical biopsy. The principal goal of this proposal is to develop markers that would assist in grading of SIL. Viral protein expression varies with stages of HPV infection and corresponding grades of SIL. Viral late proteins (L1, L2 and E4) are expressed during productive stage of infection that corresponds to LSIL. Their expression changes during carcinogenic progression to a complete absence in invasive carcinoma. Expression of late proteins in HSIL is less defined and the available data is controversial and conflicting, probably reflecting heterogeneity in histopathologic interpretation. We will use immunohistochemistry and immunofluorescence with monoclonal antibodies (both those generated in our laboratory and commercial reagents) to address: Hypothesis 1: The change in expression patterns of L1, L2, and E4 proteins in stages of HPV infection correlate with grade of SIL and can be used as biomarkers assisting with histological grading of the lesions. Specific Aim 1. To correlate expression of viral late proteins with grade of SIL in rigorously classified clinical specimens. Current dogma dictates that carcinogenic progression is linked with viral integration into the host genome such that expression of the early protein E2 is lost, but it is unclear at which stage this first occurs. Hypothesis 2: Loss of E2 expression can be detected in a fraction of HSIL, but commonly in micro- or frankly invasive disease. Specific Aim 2. We will characterize expression of E2 in HPV-associated lesions and correlate its pattern with the expression of L1, L2, E4, and grade of SIL. The variability of histopathologic grading is also highlighted by differences in diagnoses rendered on biopsies versus the definitive histologic diagnosis performed upon the excisional conization, particularly for CIN2 biopsies. Hypothesis 3: The expression of viral antigens can complement the histologic interpretation of biopsies identified as CIN2 and separate these cases into HPV infection and precancer as defined by an outcome in the excisional specimen. Specific Aim 3. We will determine correlation of L1, L2, E2 and E4 expression in HSIL 2 (CIN 2) in cervical biopsy with findings in excisional specimen and the utility of these markers as predictors of the outcome in a separate cohort of patients. HPV infection and related precancerous lesions of the cervix are common. Despite the introduction of HPV vaccines, cytologic screening programs must continue. Efforts to improve the specificity of cervical cancer screening and diagnosis are critical in the era of prophylactic HPV vaccination and call for development of new markers. This pilot study will assess the utility of immunostaining for viral protein expression as a biomarker to assist the grading precancerous cervical lesions. This study aims to lay the ground work for future prospective trials to improve the accuracy of diagnosis of precancerous HPV-related cervical lesions and thereby avoid unnecessary treatment of some patients while identifying those who need conization with greater assurance. PUBLIC HEALTH RELEVANCE: HPV infection and related precancerous lesions of the cervix are common. Despite the introduction of HPV vaccines, cytologic screening programs must continue. Unfortunately, HPV vaccination will significantly reduce the predictive value of cytologic screening. Therefore efforts to improve the sensitivity and specificity are important and call for development of new markers. This pilot study will assess the utility of immunostaining for viral protein expression as a biomarker to assist the grading precancerous cervical lesions. This study aims to lay the ground work for future prospective trials to improve the accuracy of diagnosis of precancerous HPV-related cervical lesions and thereby avoid unnecessary treatment of some patients while identifying those who need conization with greater assurance.